BNP antibody and immunoassay using it

ABSTRACT

The present invention provides antibodies for use in a method of immunoassay being antibodies specific to the polypeptide consisting of amino acids 1-76 (SEQ ID NO:1) of N-terminal brain natriuretic factor BNP(1-76). Also provided are methods and kits for the diagnosis or prognosis of conditions in which BNP(1-76) is a diagnostic or prognostic indicator, such as heart failure or hypervolemia.

This invention relates to the N-terminal section of Brain NatriureticPeptide Prohormone, BNP(1-76)(SEQ ID NO:1) and the use of antibodiesagainst this in immunoassays in biological fluids for the purpose ofbiological research and medical diagnosis, for example of heart failureor hypervolaemia.

Heart failure is a common clinical syndrome especially among elderlypeople. Population surveys indicate that the condition affects about 2%of the total population in the western world. The syndrome usuallypresents itself with an insidious onset with unspecific symptoms such asdyspnea on exertion, fatigue and peripheral oedemas. To establish thediagnosis the physician usually must either rely on his clinicalexperience or refer the patient to a cardiological center forechocardiography, radionuclide scanning, exercise testing orcatheterization.

Heart disease represents a significant drain on health resources in manymajor countries, and whilst an early diagnosis may help in controllingthe condition and preventing rapid progression to severe heart failure,it would obviously be preferable to be able to identify those patientsin which heart failure is likely to occur before it actually does so,ie. to prognose rather than diagnose.

Unfortunately, there are at present no completely satisfactory methodsfor predicting the likelihood of heart failure. Problems frequentlyobserved with such methods are insufficient accuracy and sensitivity,and the disadvantages of the necessity for expensive equipment requiringspecially trained personnel (eg. in echocardiography). A need thereforeexists for a simple method of accurately and sensitively, not onlydiagnosing, but also predicting the likelihood of onset of heartfailure.

Whilst heart failure can be defined as a symptomatic state ie. an overtdisease or syndrome, patients may frequently pass through a state ofasymptomatic cardiac dysfunction ie. a sub-clinical condition withoutovert symptoms, before heart failure manifests itself. However, we havenow found that not all patients having cardiac dysfunction go on todevelop severe heart failure, and that the risk of heart failure forsome such people is much greater than for others. To be able to identifythose people at particular risk of developing heart failure in orderthat they may be caught and treated before heart failure occurs would beof great clinical importance; at the moment existing treatments eg. ACEinhibitors are very expensive and it is not cost-effective for everyoneto be treated to try to prevent the onset of heart failure.

Brain Natriuretic Peptide (BNP) is a polypeptide originally isolatedfrom porcine brain by T. Sudoh and coworkers (Nature 1988; 332: 78-81).After cloning and sequence analysis of CDNA coding for the peptide (T.Sudoh BBRC 1989; 159: 1427-34) human BNP was shown to be produced in thehuman heart. Human Brain Natriuretic Peptide is believed to be producedin cardiac myocytes as a prohormone (proBNP or BNP(1-108)). proBNPconsists of 108 amino acids and is split, before or during secretion, atamino acids Arg76--Ser77 into BNP and the N-terminal part of theprohormone, BNP(1-76)(SEQ ID NO:1), that is the peptide consisting ofthe first 76 amino acids from the N-terminal of proBNP.

The BNP(77-108) plasma concentration is increased in patients sufferingfrom heart disease leading to heart failure. The cardiac monocytessecrete another factor, namely atrial natriuretic factor (ANF) but thesecretory response to heart failure or incipient heart failure seems tobe much larger in the BNP system compared to the ANF system (Mukoyama etal, J Clin Invest 1991; 87: 1402-12).

The present invention is based on the concept that human BNP(1-76)(SEQID NO:1), due to a long half-life as compared with BNP hormone itselfand high initial concentration, is a particularly good diagnosticindicator or predictor of heart disease and also of hypervolaemia.

Human BNP(1-76)(SEQ ID NO:1) may thus be used to provide the basis ofeither a diagnostic or a prognostic test for heart failure, primarily inthe biosynthesis of antibodies for use in such a test but also ascompeting antigen in competitive binding immunoassays. For such use inmaking antibodies BNP(1-76)(SEQ ID NO:1) or an antigenic fragmentthereof may advantageously be conjugated to an immunogenic protein orpeptide such as PPD, a protein derivative of tuberculin, Keyhole LimpetHaemocyanin or bovine serum albumin.

Thus human BNP(1-76)(SEQ ID NO:1) or an antigenic fragment thereof orpolypeptide extension thereof lacking BNP activity and having at leastone antigenic epitope of human BNP(1-76)(SEQ ID NO:1), conjugated to oneor more immunogenic polypeptides, constitutes one aspect of the presentinvention; these polypeptides may be used to make either polyclonal ormonoclonal antibodies specific to BNP(1-76)(SEQ ID NO:1). Suchmonoclonal and polyclonal antibodies constitute two further aspects ofthe invention.

According to a still further aspect of the invention we provide a methodof immunoassay for human BNP(1-76)(SEQ ID NO:1) or an antigenic fragmentthereof or polypeptide extension thereof lacking BNP activity whereinthe primary binding partner therefor is a monoclonal or polyclonalantibody according to the invention. Methods of immunoassay are ofcourse well known in the art eg. RIA, ELISA, fluorescence immunoassay(FIA) or dry chemistry test strip immunoassays. Such an immunoassaywill, in general, use a monoclonal or polyclonal antibody according tothe invention in immobilised form, e.g. on microtitre plates, membranesor beads, to isolate the target BNP(1-76) compound. In a sandwich assay,the bound antigen may be labelled using additional soluble antibodyaccording to the invention, which may be monoclonal or polyclonal andwhich may either carry a label or, more conveniently, may itself belabelled subsequently by reaction with a secondary antibody carrying alabel.

Thus, if the primary antibody according to the invention is raised inmice or rabbits, the labelled secondary antibody may be an anti-mouse oranti-rabbit antibody.

Suitable labels include radionucleides, fluorescent substances eg.europium based fluorogens, enzymes, for example as used in ELISA systemsemploying automated hybrid methods or dyes or coloured particles such ascolloidal gold.

Alternatively, a competitive binding assay may be used, wherein a knownquantity of labelled human BNP(1-76)(SEQ ID NO:1), or antigenic fragmentor inactive extension thereof, is added to the analyte solution andcontacted with a limited quantity of the immobilised monoclonal orpolyclonal antibody, whereby the amount of labelled antigen which isimmobilised is inversely proportional to the amount of target antigenpresent in the analyte.

The invention thus extends to labelled forms of human BNP(1-76)(SEQ IDNO:1) or antigenic fragments or polypeptide extensions thereof lackingBNP activity and to labelled forms of the antibodies of the invention.

The invention also comprises a kit for immunoassay of humanBNP(1-76)(SEQ ID NO:1) or an antigenic fragment or polypeptide extensionthereof lacking BNP activity comprising:

(a) a monoclonal or polyclonal antibody according to the invention inimmobilised form and, at least one further component selected from;

(b) a labelled sample of BNP(1-76)(SEQ ID NO:1) or an antigenic fragmentor polypeptide extension thereof lacking BNP activity;

(c) said monoclonal or polyclonal antibody in non-immobilised form;

(d) a labelled secondary antibody specific to said antibody (c).

Such an immunoassay and kit may be used in research into relatedbiological systems as well as for diagnosis or prognosis of conditionswherein the human BNP(1-76)(SEQ ID NO:1) level in body fluids is adiagnostic or predictive indicator.

The invention also comprises a method of diagnosis or prognosis of acondition in which the concentration of human BNP(1-76)(SEQ ID NO:1) oran antigenic fragment or polypeptide extension thereof lacking BNPactivity is a diagnostic or predictive indicator, wherein a body fluidof a patient is subjected in vitro to immunoassay to detect or assay thepresence or quantity therein of human BNP(1-76)(SEQ ID NO:1).

We have recently found that another natriuretic factor namely pro-ANF,and in particular N-terminal pro-ANF, can serve as an indicator of riskof heart failure in patients lacking overt symptoms of heart failure.The level of pro-ANF in body fluids can be directly related to the riskof heart failure, predominantly related to increased atrial pressure. Incontrast, BNP(1-76)(SEQ ID NO:1) is predominantly an indication of aheart condition related to increased ventricular pressure. HumanBNP(1-76)(SEQ ID NO:1) as an antigenic fragment or inactive polypeptideextension thereof can also be used to assess risk of heart failure inaddition to its use in diagnosis of actual heart failure. Furthermore,assay of both N-terminal pro-ANF and BNP(1-76)(SEQ ID NO:1) in bodyfluids can assist in determining whether atrial or ventricular pressureis concerned.

Thus, the immunoassay can be used in the monitoring of heart failuretreatment. Such treatment is aimed at reducing the hypervolemia andexcessive vasoconstriction seen in heart failure by the administrationof diuretics and vasodilators. By decreasing the pressure in the cardiacchambers such treatment will lower cardiac production of BNP(1-76)(SEQID NO:1). The resultant decrease in plasma BNP(1-76) concentration serveto inform the physician of a significant drug effect. On the contrary,an increase in plasma BNP(1-76) indicates that an adjustment of dosagemight be necessary.

Although less well documented at this time human BNP(1-76)(SEQ ID NO:1)may also be used as a diagnostic tool in the diagnosis of hypervolemiawithout heart failure. The immunoassay therefore has potential use alsoin the inhospital intensive care setting where monitoring of volumestatus is essential.

The body fluid on which the immunoassay is performed may be any bodyfluid in which the human BNP(1-76)(SEQ ID NO:1) is located, butconveniently will be plasma or serum. In some cases it may be convenientto extract the peptide, or otherwise treat the sample prior to assay.

The human BNP(1-76)(SEQ ID NO:1) peptide or an antigenic or immunogenicfragment thereof may be produced by synthesis from its constituent aminoacids or by assembly of pre-synthesised blocks of amino acids usingtechniques well known in the art. Where labelled material is required,the label may be introduced by standard techniques.

For the purpose of raising monoclonal or polyclonal antibodies, thehuman BNP(1-76)(SEQ ID NO:1) or antigenic fragment thereof may beconjugated to an immunogenic protein or peptide, for example PPD, aprotein derivative of tuberculin, eg. using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide according to the method ofStaros et al (Analyte Biochem 1986; 156: 220-222).

The antibodies of the invention may be made by injecting a host animal,eg. a mouse or rabbit, with the BNP antigen of the invention,advantageously a conjugate with an immunogenic protein as describedabove, to provide either a serum containing polyclonal antibodies orspleen cells for conversion to hybridomas or immortalised cell linesproducing monoclonal antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Examples are given by way of illustration only withreference to the accompanying drawing in which:

FIG. 1 shows a standard curve for immunoassay for BNP(1-76) usingsynthetic BNP(47-64) as immunogen, standard and tracer, and polyclonalrabbit antibody. (Abscissa shows BNP(47-64) pmol/l; ordinate shows %binding (B/B(O))).

EXAMPLE 1 Production of Monoclonal Antibody Against BNP(1-76)

1) Conjugation

Three synthesized fragments of BNP(1-76)(SEQ ID NO:1): BNP(1-21),BNP(22-46) and BNP(47-64) were acquired from Peninsula laboratories andconjugated to PPD (protein derivative of tuberculin) according to Staroset al (Analyt Biochem 1986; 156: 220-222).

2) Immunization

Balb C mice, preimmunized with BCG antigen were utilized. The micereceived a 50 microgram mixture of the three conjugates in 200 μl ofFreunds incomplete adjuvant. The mixture was given in 2×200 μlinjections on 2 occasions 2 weeks apart. 2 weeks after the lastinjection 50 μg of conjugate mixture in saline was injectedintraperitoneally.

3) Fusion

3 days after intraperitoneal immunization mouse splenic cells were fusedwith SP 2/0 myeloma cells and the resultant hybridomas selected in HATmedium. The suspension of hybridomas was distributed in 960 wells inDulbeccos medium enriched with 10% human endothelial cell supernatant.

4) Screening

Method 1

Costar microtiter plates were coated with a mixture of the synthetic BNPpeptide sequences (0.5 μg/ml). Supernatants were then added and bindingof antibody from supernatants was screened by ELISA through the additionof anti mouse IgG conjugated to horseradish peroxidase enzyme followedby substrate solution (OPD).

Method 2

An alternative method of screening is to coat Greiner microtiter plateswith goat anti mouse IgG (1.0 μg/ml). Supernatants are then added andincubated. Biotinylated synthetic BNP peptide sequences are added andthe ability of supernatants to bind peptide are screened by ELISAthrough the addition of streptavidin-conjugated horseradish peroxidaseenzyme followed by substrate solution (OPD).

5) Cloning

Hybridomas producing antibodies to the peptide mixture were cloned andsubcloned in two runs. Clone 1C7 was shown to react with peptidesequence BNP(47-64). This clone was grown and the supernatant utilisedin immunoassay for BNP(1-76).

EXAMPLE 2 Immunoassay for BNP(1-76)(SEQ ID NO:1)

The 1C7 antibody can be utilised in various types of immunoassays forBNP(1-76)(SEQ ID NO:1). These include

a) Radioimmunoassay (RIA)

b) Europium Fluorescence immunoassays (FIA)

c) Enzyme linked immunosorbent assays (ELISA) including automated hybridmethods running on micro titer plates or membranes

d) Various dry-chemistry test strip immunoassays

The following is an example of a sandwich ELISA.

Costar microtiter plates are precoated with the 1C7 antibody. Sample orstandard is added to the wells and after 2 hours of incubation the wellsare washed and a secondary antibody (polyclonal or monoclonal) towardsBNP(1-76)(SEQ ID NO:1) is added. Again after 2 hours a horseradishperoxidase-labelled anti mouse (rabbit) antibody is supplied and finallyafter the addition of O-phenylene-diamine substrate the colour is readin a platereader.

EXAMPLE 3 Immunoassay for BNP(1-76) Utilizing Polyclonal Rabbit Antibody

A synthetic peptide subsequence of BNP(1-76)(SEQ ID NO:1), in this caseBNP(47-64), was conjugated to PPD according to Staros et al., (Supra).Rabbits were BCG vaccinated and then repeatedly immunized with theconjugated peptide.

Iodination (¹²⁵ I) of synthetic BNP(47-64), with a tyrosine group addedat the N-terminal end, was done by the chloramine-T method as follows:

Chloramine-T Method

1) 5 μg of the synthetic peptide was reconstituted with 20 μlsodiumphosphate buffer (0.25M, pH 7.5).

2) Approximately 5 μl of ¹²⁵ -I was added (0.5 mCi).

3) 5 μl of chloramine-T (1 mg/ml) was added and incubated for 45seconds.

4) 5 μl of sodiummetabisulphite (1 mg/ml) was added and incubated for 45seconds.

5) The mixture was then fractionated on a column with Sephadex G10.

6) The fractions were counted with a gamma-counter, and thefraction/fractions with highest counts per minute (cpm) were selected astracer for use in the RIA methods.

Sample or standards (BNP 47-64!), together with tracer (iodinatedBNP(47-64)) and polyclonal antibody from rabbit serum, are mixed inpolystyrene assay tubes. After 48 hours of incubation at 4° C., normalserum from rabbit, and goat anti-rabbit IgG are added. After 2 hours ofincubation, polyethyleneglycol (PEG) is added and the samples arecentrifuged. The supernatant is removed and the counts per minute (cpm)in precipitate are measured with a gammacounter. An example of astandard curve obtained by this type of assay is shown in FIG. 1.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 76 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       HisProLeuGlySerProGlySerAlaSerAspLeuGluThrSerGly                              151015                                                                        LeuGlnGluGlnArgAsnHisLeuGlnGlyLysLeuSerGluLeuGln                              202530                                                                        ValGluGlnThrSerLeuGluProLeuGlnGluSerProArgProThr                              354045                                                                        GlyValTrpLysSerArgGluValAlaThrGluGlyIleArgGlyHis                              505560                                                                        ArgLysMetValLeuTyrThrLeuArgAlaProArg                                          657075                                                                        __________________________________________________________________________

I claim:
 1. An antibody composition comprising an antibody whichspecifically binds to a polypeptide consisting of amino acids 1-76 ofthe N-terminal of human pro-brain natriuretic factor (BNP(1-76)(SEQ IDNO:1)).
 2. The antibody composition as claimed in claim 1 whichcomprises a monoclonal antibody.
 3. The antibody composition as claimedin claim 1 which comprises a polyclonal antibody.
 4. The antibodycomposition as claimed in claim 1 carrying a label.
 5. The antibodycomposition as claimed in claim 4 in which the label is a radionuclide,a fluorescent substance, an enzyme, a dye or coloured particles.
 6. Theantibody composition of claim 1, wherein the antibody is immobilized ona solid support.
 7. A method of immunoassay for BNP(1-76), (SEQ IDNO:1), which comprises:(a) contacting a body fluid from a patient with aprimary antibody according to claim 1 to form a primaryantibody-BNP(1-76)(SEQ IS NO:1) complex; and (b) detecting the formationof said complex.
 8. The method as claimed in claim 7 in which theprimary antibody is immobilized on microtitre plates, membranes orbeads.
 9. The method as claimed in claim 8 in which said detectingcomprises contacting said complex with a second antibody which bindsBNP(1-76)(SEQ ID NO:1) to form a second complex comprising a primaryantibody-BNP(1-76)(SEQ ID NO:1)-secondary antibody and detecting theformation of said second complex to provide a sandwich assay.
 10. Themethod as claimed in claim 7 wherein said primary antibody isimmobilized and wherein said contacting comprises first adding a knownquantity of labelled human BNP(1-76)(SEQ ID NO:1) or a labelledantigenic fragment thereof to the body fluid in solution to provide acompetitive binding assay.
 11. A labelled human BNP(1-76)(SEQ ID NO:1)or a labelled antigenic fragment thereof.
 12. A kit for immunoassay ofhuman BNP(1-76)(SEQ ID NO:1) lacking BNP activity comprising:(a) anantibody according to claim 1 in immobilized form and, at least onefurther component selected from the group consisting of: (b) a labelledsample of human BNP(1-76)(SEQ ID NO:1) or an antigenic fragment thereoflacking BNP activity; (c) an antibody according to claim 1; and (d) alabelled secondary antibody which specifically binds to an antibodyaccording to claim
 1. 13. An in vitro method of diagnosis or prognosisof heart disease comprising:(a) contacting a body fluid from a patientwith a primary antibody according to claim 1 to form a primaryantibody-BNP(1-76)(SEQ IS NO:1) complex; and (b) detecting the formationof said complex to determine the level of BNP(1-76)(SEQ ID NO:1) in thebody fluid wherein an increased level of BNP(1-76)(SEQ ID NO:1) ascompared to normal individuals is indicative of a diagnosis or prognosisof heart disease.